The long-term goal of this research is to understand the folding and structural assembly of multiple repeat proteins. Recent progress in genomic sequencing analysis indicates that as much as 30% of all proteins are composed of modular, internal repeat units. These proteins often hold important regulatory functions using the repeating units as recognition modules to interact with a wide range of protein or non-protein targets. The internal repeat proteins also provide us with a unique opportunity to study the fundamental principles of protein folding because the regular, periodic structure and the dominance of short-range interactions greatly simplify the topology of the intramolecular coupling network. Among all internal repeat proteins, those containing the ankyrin repeat, a 33-residue sequence motif, form one of the best-characterized and largest families. Specific aims of this grant include the use of NMR-based urea denaturation and hydrogen exchange studies to investigate the folding mechanism of myotrophin, a cardiomyogenic hormone consisting of four ankyrin repeats. Myotrophin was chosen as a model system because it is well-folded and soluble under a broad range of conditions. The goal of this study is to identify the folding initiation sites and to provide a residue-specific picture of the folding process. To further simplify the complexity of molecular interactions, a series of consensus ankyrin repeat proteins with identical repeats have been designed and characterized. It is anticipated that studies of these proteins by urea denaturation, fluorescence labeling, and limited proteolysis will eventually allow us to build a theoretical model on the folding of ankyrin repeats. Finally, the rules of ankyrin repeat based molecular recognition will be probed using the consensus ankyrin repeat proteins as a scaffold to design specific binding interactions. Taken together, these combined approaches will generate important information regarding the folding of multiple repeat proteins and how the structural and folding abnormalities may underline serious human diseases.